Intracellular magnesium optimizes transmission efficiency and plasticity of hippocampal synapses by reconfiguring their connectivity

Synapses at dendritic branches exhibit specific properties for information processing. However, how the synapses are orchestrated to dynamically modify their properties, thus optimizing information processing, remains elusive. Here, we observed at hippocampal dendritic branches diverse configurations of synaptic connectivity, two extremes of which are characterized by low transmission efficiency, high plasticity and coding capacity, or inversely. The former favors information encoding, pertinent to learning, while the latter prefers information storage, relevant to memory. Presynaptic intracellular Mg2+ crucially mediates the dynamic transition continuously between the two extreme configurations. Consequently, varying intracellular Mg2+ levels endow individual branches with diverse synaptic computations, thus modulating their ability to process information. Notably, elevating brain Mg2+ levels in aging animals restores synaptic configuration resembling that of young animals, coincident with improved learning and memory. These findings establish intracellular Mg2+ as a crucial factor reconfiguring synaptic connectivity at dendrites, thus optimizing their branch-specific properties in information processing.


Supplementary
Average q of synapses at dendritic branches GluA2, PSD95 IF

• Serial studies regarding the role of Mg 2+ on brain health and aging
As this study is part of a series investigating the positive impact of Mg 2+ on brain health and aging, we would like to provide a concise overview of our serial studies, which explore the role of Mg 2+ ions in synaptic, neuronal, circuitry, and cognitive functions over the years.Our investigations span from in vitro experiments to in vivo studies involving animals and humans, from the microlevel of proteins and single synapses to the macrolevel behaviors and cognitive functions.
Initially, we observed that elevating extracellular Mg 2+ concentration enhanced long-term potentiation (LTP) of synapses in cultured hippocampal neurons, leading to increased expression of GluN2B-containing NMDARs 1 .Building upon this discovery, we hypothesized that raising brain Mg 2+ levels could improve synaptic plasticity in the hippocampus, thereby enhancing cognitive functions, especially learning and memory, in intact animals.To achieve this, we developed Magnesium L-Threonate (MgT), a compound that effectively increased Mg 2+ bioavailability in the cerebrospinal fluid (CSF) when orally consumed 2 .Elevating Mg 2+ in the rodent brain's CSF demonstrated enhanced synaptic plasticity and cognitive functions in both young and aging animals 2 , validating our in vitro hypotheses.Concurrently, we observed beneficial effects in treating cognitive declines in Alzheimer's disease model mice 3 and depression model mice 4 .
Encouraged by these animal studies, we expanded our research to translational studies.The first double-blind placebo-controlled clinical study demonstrated that MgT supplementation improves cognitive functions in mild cognitive impairment (MCI) patients 5 .Currently, three ongoing FDAapproved phase 2b/3 clinical trials are investigating MgT's role in treating cognitive disorders in humans, including Alzheimer's disease 6 , Attention Deficit Hyperactivity Disorder (ADHD) 7 , and depression/anxiety.Despite these promising clinical studies, the mechanism underlying the powerful impact of Mg 2+ on human brain functions remains elusive.Initially, we believed that the primary effect of extracellular Mg 2+ targets NMDARs to influence plasticity based on electrophysiological and molecular evidence 1 , demonstrating its extracellular modulatory effect.However, we later discovered that the beneficial effects extend beyond modulating synaptic plasticity.Subsequently, our findings revealed that intracellular Mg 2+ plays an even more crucial role in regulating the density of functional presynaptic boutons 8 , offering a new perspective on Mg 2+ 's role in promoting brain health.
Intriguingly, in the compound MgT, threonate (T) itself synergizes with Mg 2+ , elevating intracellular Mg 2+ levels and increasing the density of presynaptic boutons in cultured hippocampal neurons 9 .This insight contributes to understanding the pharmacological effects of MgT in elevating brain Mg 2+ levels and enhancing animal cognitive functions.Despite focusing on single synapses in these mechanistic studies, it remains unclear how intracellular Mg 2+ governs multiple synapses along individual dendritic branches, imparting different transmission efficiency, plasticity, and coding capacity.Given the fundamental role of dendritic branches in processing information, addressing this question could illuminate how nearby synapses are regulated to achieve specific computational features at individual dendritic branches and identify endogenous factors controlling such synaptic organization.

• The concept of synaptic configuration
In the current article, the landing point is to tackle a longstanding question in the field: how nearby synapses at individual dendritic branches are organized to generate distinct synaptic computations, essentially regulating the "transfer function" of synapses at a dendritic branch.This question is crucial as dendritic branches are considered the basic computational unit for information processing underlying cognitive functions.Our findings reveal that intracellular Mg 2+ serves as an endogenous factor in organizing nearby synapses from different presynaptic neurons, influencing the configuration of synaptic connectivity at individual dendritic branches.This, in turn, determines the "transfer function" of each dendritic branch.We introduced a general principle of synaptic organization at dendritic branches, proposing that nearby synapses are consistently organized along an individual branch to maintain a constant total presynaptic strength (the first part of the Discussion).
It's important to note that the concept of configuration is more generalized, with the regulatory effect of intracellular Mg 2+ serving as a significant example.As different configurations impart distinct features of synaptic computations to an individual branch, the transition between configurations becomes crucial for branch-specific synaptic computations during information processing for learning and memory.Significantly, our principle hints at the possibility of other essential endogenous factors, beyond intracellular Mg 2+ , regulating synaptic configuration.Such factors could be promising candidates for anti-brain aging and anti-neurodegeneration strategies, providing a novel avenue for drug exploration.Overall, we believe that this study offers precise and comprehensive mechanisms, serving as a cornerstone in our series of studies on the beneficial effects of brain Mg 2+ in maintaining brain health.
• Rationales for the experimental Mg 2+ condition Mg 2+ stands as the second most abundant intracellular mineral after K + and is present in substantial amounts in the cerebrospinal fluid (CSF) of both rodents (around 0.8 mM) and humans (around 1.0-1.2mM in healthy individuals) (for a review 10 ).The concentrations of 0. • Aging is a risk for Mg 2+ deficits Aging poses a significant risk for Mg 2+ deficit, as highlighted in various reviews 10,[14][15][16][17][18][19][20][21] .Clinical studies reveal a substantial decrease in brain cerebrospinal fluid (CSF) Mg 2+ concentration during aging and neurodegenerative diseases in humans 22 .Notably, elemental Mg 2+ levels are markedly reduced in the brains of Alzheimer's disease patients 23,24 .As regard to intracellular Mg 2+ levels, clinical studies employed the phosphorus magnetic resonance spectrum ( 31 P MRS), a method for measuring intracellular ionized Mg 2+ concentrations in vivo, demonstrate a significant decrease in body [Mg 2+ ]i during aging 25,26 .These findings suggest that the decline in [Mg 2+ ]i serves as a hallmark of aging and neurodegeneration, emphasizing the crucial role of Mg 2+ in protecting brain health.Indeed, both animal and human studies underscore the effectiveness of brain Mg 2+ supplementation in addressing cognitive deficits associated with aging and neurodegenerative disorders.
In animal studies, brain Mg 2+ supplementation exhibits a protective effect against aging-dependent cognitive declines 10,27 .Our research demonstrates that cognitive impairments in aged animals 2 and Alzheimer's disease model animals 3 can be significantly ameliorated through brain Mg 2+ supplementation.Additionally, brain Mg 2+ supplementation shows promise in treating other neurodegenerative diseases.Independent studies report that MgT treatment effectively alleviates motor deficits and dopamine neuron loss in a mouse model of Parkinson's disease 28 .
Translational research assesses the efficacy of MgT (also known as L-threonic acid magnesium salt, L-TAMS) treatment in ameliorating cognitive deficits related to aging and neurological disorders.In our initial double-blind, placebo-controlled clinical study, MgT supplementation is shown to significantly reverse age-dependent cognitive impairment 5 .Consistent results are reproduced in other double-blind, placebo-controlled clinical studies conducted by independent groups 29 .Moreover, a clinical trial by Stanford University researchers demonstrates that MgT treatment effectively alleviates cognitive decline in Alzheimer's disease patients 30 .Another open-label pilot study at Massachusetts General Hospital reports that MgT treatment improves cognitive functions in ADHD patients 7 .
Recently, the World Health Organization reached a consensus that dietary Mg 2+ intake is lower than recommended in a majority of the world's population, especially in the aging demographic (https://www.who.int/publications/i/item/9789241563550; see also clinical trials 31,32 ).Therefore, based on the compelling evidence, elevating brain Mg 2+ levels in the elderly emerges as a promising strategy to minimize, or even prevent, aging-dependent cognitive deficits.

• Implications of Mg 2+ deficits for brain aging
Over the past decades, numerous animal and clinical studies have extensively documented progressive deficits in body Mg 2+ levels during aging, likely stemming from insufficient intake and disorders in Mg 2+ metabolism (for reviews [14][15][16][17][18][19][20] ).However, the underlying mechanisms still require in-depth exploration.Mg 2+ deficiency emerges as a high-risk factor for brain aging and neurodegeneration, crucial for sustaining brain health in both young and aged animals.
Firstly, Mg 2+ sufficiency proves pivotal for maintaining brain health in young adults.On one hand, a 30-35% reduction in dietary Mg 2+ causes a 40% decrease in [Mg 2+ ]i in the brains of young adult animals 33 , leading to significant impairments in cognitive functions, especially hippocampusdependent learning and memory (for examples see Refs [34][35][36] ).Moreover, dietary Mg 2+ deficiency induces systemic low-grade neuroinflammation in young adults, a hallmark of aging and neurodegenerative diseases (for a review 37 ).On the other hand, an early study reported that chronic feeding of a high-Mg 2+ diet (2% elemental Mg 2+ in the diet) increases brain Mg 2+ levels and improves learning behaviors in young rats 38 .Consistently, our studies have demonstrated that when young animals consume a normal-Mg 2+ diet, supplementation of brain Mg 2+ through oral intake of MgT in drinking water further enhances their learning and memory 2 .
Secondly, Mg 2+ supplementation reverses cognitive declines in aging and neurodegeneration.Early studies have reported an improvement in cognitive functions in aged animals through a high dosage of Mg 2+ in the diet 38 .Our previous studies show restored learning and memory in aged rats by elevating brain Mg 2+ levels through MgT treatment 2 .Additionally, we demonstrate that cognitive declines can be effectively ameliorated by MgT treatment in Alzheimer's disease model mice (APP/PS1 transgenic mice) 3 .Consistently, an independent study indicates that MgT treatment can reduce neuroinflammation and alleviate cognitive decline in APP/PS1 transgenic mice 39 .
Overall, converging evidence suggests a crucial role of Mg 2+ in maintaining brain health in young adults and during brain aging.

Fig. 4 |SupplementaryFig. 5 |
Calibration of intracellular Mg 2+ concentrations.a, Representative confocal images to show MgGrn fluorescence signals with various [Mg 2+ ]i in boutons.Ionophore was used to equilibrate various concentrations of [Mg 2+ ]i.Following the collection of confocal images of MgGrn, FM4-64 labeling elicited by 600 APs at 10 Hz field simulation was utilized to visualize the boutons and normalize the bouton volume (see Methods).b, Calibration curve (n = 6 biological repeats for each concentration) that was fitted by the Hill equation (R 2 = 0.99, Kd = 0.91 mM).Note the quasi-linear relationship between MgGrn fluorescence and real [Mg 2+ ]i within the range of 50-1200 f.u.(fluorescence unit).Blue line/error band, fitted curve/95% CI.Source data are provided as a Source Data file.Supplementary Fig. 5 | See next page for caption.Measurement of Pr and evoked presynaptic Ca 2+ influx in single boutons a, Experimental design.Left, schematic to show FM5-95 labeling in the boutons transfected by CaMKIIα-Synaptophysin-GCaMP6f (SypGCaMP6f).

Supplementary Fig. 6 | Labeling of released vesicles and presynaptic proteins in the same boutons. a, Schematic to
-) boutons (n = 406, 232 boutons from 5 repeats).Inset, discrete data points in violin plots, where black and magenta lines indicate median and quartiles.Two-sided Kolmogorov-Smirnov test, P = 0.86 (NS).c, Left, average traces of Ca 2+ influx of boutons (visualized by SypGCaMP6f) evoked by various input patterns (n = 302, 387 boutons from 5, 5 repeats).Traces were averaged from 30 sweeps of the boutons.Right, relationship between [Ca 2+ ]evoke and AP number (n = 5 repeats).Solid lines, linear regressions.Dashed line, extension of the black line.The frequency of APs in all bursts was 100 Hz. d,
8-1.2 mM used in the current study are supported by multiple lines of evidence.Under in vivo conditions, [Mg 2+ ]o in the CSF of animal brains can increase by 21% above control (i.e., from ~1 mM to 1.2 mM) 5.5 hours after intravenous injection of MgCl2 or MgSO4 (Ref 11 ).